Tuberculosis (TB) is the primary cause of death due to a single microbial pathogen, accounting for 2 to 3 million deaths and an estimated 8 to 10 million new cases per year worldwide (1). It is estimated that one third of the world's population is infected with Mycobacterium tuberculosum. The global epidemic is growing, concomitant with HIV co-infection and the increasing prevalence of drug resistant strains. Genotyping clinical isolates of M tuberculosum has the potential to significantly impact both clinical practice and public health. Clinically, determining strain relationships can distinguish treatment failure versus potential laboratory cross-contamination, and reactivation of drug-resistant disease versus infection with another strain (24). In the public health arena, determining strain relationships during the course of a tuberculosis outbreak can focus public health resources. However, the most discriminatory M tuberculosum genotyping method, IS6110 restriction fragment length polymorphism (RFLP) analysis, is time-consuming, technically demanding, and difficult to standardize between laboratories (3). In addition, IS6110 RFLP does not provide sufficient strain discrimination when fewer than six IS6110-hybridizing bands are present. A secondary genotyping method is required in these instances. A number of PCR-based genotyping methods have been developed, but are limited in their ability to reproducibly provide a level of strain discrimination that matches associations defined by traditional epidemiology. In addition, the high degree of coding region sequence conservation between strains of M tuberculosum virtually excludes the use of more commonly used population genetics methods like multi-locus enzyme electrophoresis, restriction fragment length end labeling analysis, and gene sequencing (12).
Spoligotyping is currently the most widely used PCR-based method of M. tuberculosum genotyping due to the relative technical ease of the protocol and the ability to compare results between laboratories using a standardized nomenclature system (6). However, the discriminatory power of this method is low. In a database of 3,319 spoligotypes from isolates collected in geographically distinct areas, seven spoligotype patterns were found for 37% of all clustered isolates (18). These spoligotype patterns are associated with strains having a much larger variety of IS6110 RFLP patterns. For instance, 18% of isolates in the spoligotype database had the “Beijing” spoligotype pattern, characterized by the presence of spacer oligonucleotides 39 through 43 (18). However, although the W-Beijing family of strains is recognized as one of the most homogeneous families of M. tuberculosum strains (19), the Beijing spoligotype is associated with at least 450 distinct strains as determined by IS6110 RFLP analysis (2).
Following the public release of the complete genomic sequences of M. tuberculosum strains H37Rv and CDC-1551, investigators began the process of identifying repetitive genetic elements to develop higher resolution DNA typing systems. Variable number tandem repeats (VNTR) of 21-111 bp genetic elements have been identified (9, 14, 17, 20, 21). A set of 12 VNTRs, also known as mycobacterial interspersed repeat units (MIRUs), was more discriminatory than IS6110 RFLP when used to analyze 180 M. tuberculosum isolates with 6 or fewer IS6110-hybridizing bands. The combination of MIRU analysis and spoligotyping and IS6110 RFLP analysis has provided maximum specificity (5).
However, a greater discriminatory power for resolution and differentiation of species is necessary for sub-typing Mycobacterium tuberculosum species in order to track sources of infection and ultimately to prevent the spread of disease. Methods and means for determining the genetic differences between M. tuberculosum species with speed, accuracy and with great discriminatory capacity have been sought.